Method of Treating Hepatic Steatosis

ABSTRACT

A method of treating hyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulin insensitivity), impaired glucose tolerance, high glucose levels, pulmonary hypertension, and/or a condition arising from any of the foregoing in a patient is provided. The method comprises knocking down mARC2 or mARC1 expression in the patient, or otherwise decreasing mARC2 and mARC1 activity in the patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/072,468 filed Oct. 16, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/202,667 filed Nov. 28, 2018, which issued asU.S. Pat. No. 10,835,581 on Nov. 17, 2020, which claims the benefit ofU.S. Provisional Patent Application No. 62/591,390 filed Nov. 28, 2017,which is incorporated herein by reference in its entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 6527_2005343_ST25.txt. The size ofthe text file is 14,554 bytes, and the text file was created on Sep. 17,2020.

Provided herein are methods of treating obesity, hyperglycemia, insulinresistance, impaired glucose tolerance, diabetes, and metabolic syndromein a patient, and related compositions.

Hyperglycemia is a condition in which excess glucose circulates inblood. The consequences of hyperglycemia has been associated withcomorbidities including cardiovascular disease, vision impairment,various forms of neuropathy and cognitive impairment, stroke, andperipheral vascular disease. The common therapeutic approach, inaddition to major modifications in an individual's dietary nutrition andphysical activity, includes the use of anti-hyperglycemic drugs andinsulin. Hyperglycemia is chronic and progressive, and, to date, notreatment is able to reverse the progression, there remains a need foran improved medicament for treating hyperglycemic conditions, such asinsulin resistance, impaired glucose tolerance, diabetes, and metabolicsyndrome in a patient, and related compositions, as well as treatmentfor obesity.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant No.HL103455, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SUMMARY

A method of treating hyperglycemia, diabetes, metabolic syndrome,insulin resistance (insulin insensitivity), impaired glucose tolerance,high glucose levels, pulmonary hypertension, and/or a condition arisingfrom any of the foregoing in a patient is provided. The method comprisesknocking down expression of mARC2 in a patient, thereby treatinghyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing in the patient.

Also provided is a method of treating hyperglycemia, diabetes, metabolicsyndrome, insulin resistance (insulin insensitivity), impaired glucosetolerance, high glucose levels, pulmonary hypertension, and/or acondition arising from any of the foregoing in a patient. The methodcomprising knocking down expression of mARC1 in a patient, therebytreating hyperglycemia, diabetes, metabolic syndrome, insulin resistance(insulin insensitivity), impaired glucose tolerance, high glucoselevels, pulmonary hypertension, and/or a condition arising from any ofthe foregoing in the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides an exemplary nucleic acid sequence for a human mARC2mRNA cDNA (NM_001317338.1, Homo sapiens mitochondrial amidoxime reducingcomponent 2 (mARC2), transcript variant 1, mRNA, (SEQ ID NO: 1)). FIG.1B provides an exemplary nucleic acid sequence for a human mARC1 mRNAcDNA (NM_022746.3, Homo sapiens mitochondrial amidoxime reducingcomponent 1 (mARC1), mRNA (SEQ ID NO: 3)). FIG. 1C provides the fulllength human mARC 2 protein sequence (NP_001304267.1, Homo sapiensmitochondrial amidoxime reducing component 2 precursor protein, 335amino acid protein, (SEQ ID NO: 2). FIG. 1D provides the full-lengthhuman mARC 2 protein sequence (NP_073583.3, Homo sapiens mitochondrialamidoxime reducing component 1 precursor protein, 337 amino acidprotein, (SEQ ID NO: 4).

FIGS. 2A-2C. Deleting mARC2 in mice prevents age-associated weight gain.(FIG. 2A) Photograph of 12-month old male mARC2 KO and WT mice. (FIG.2B) relative mARC2 transcript levels in mouse liver measured by qRT-PCR.(FIG. 2C) mARC2 protein levels in liver homogenates measured withwestern blot. mARC2 KO, C57BL/6N mARC2^(tm2A). WT, wildtype. N=4-5 pergroup. Students t-test used to calculate p-value.

FIGS. 3A-3D: Deleting mARC2 in moderately aged (10-month-old) male micegenerates differences in body composition (BC). (FIG. 3A) Body weight(BW). (FIG. 3B) fat mass, measured by proton NMR. (FIG. 3C) lean massnormalized to BW. (FIG. 3D) fat mass normalized to BW. N=8 per group.Unpaired t-test used to calculate significance. ***, p-value<0.001. **,p-value<0.01.

FIGS. 4A-4D: Metabolic cage data collected with individually housedmoderately aged (10-month-old) male mice. Energy expenditure normalizedto BW— (FIG. 4A) raw data collected over 24 hours, (FIG. 4B) summary ofA. Total activity—(FIG. 4C) raw data collected over 24 hours and (FIG.4D) summary of C. Total activity is defined as the total number of beambreaks recorded over a 1 min interval. N=8 per group. Unpaired t-testused to calculate significance.

FIGS. 5A and 5B: Plasma (FIG. 5A) glucose and (FIG. 5B) insulin inlevels 10-month-old) male mice after an overnight fast. N=8 per group.Unpaired t-test. **, p-value<0.01.

FIGS. 6A-6D: Plasma glucose and insulin levels post-intraperitonealglucose challenge in housed moderately aged (10-month-old) male mice.(FIG. 6A) Glucose levels over time. (FIG. 6B) glucose clearance, areaunder the curve (AUC) from A. (FIG. 6C) Insulin levels over time. (FIG.6D) Insulin clearance, from AUC of data in FIG. 6C. N=8 Unpaired t-test.***, p-value<0.001. **, p-value<0.01. *, p-value<0.05.

FIG. 7: Histology (H&E staining) of paraffin embedded liver fromlittermate 12-month-old WT and mARC2 KO male mice feed normal chow.

FIGS. 8A-8B: Plasma glucose (FIG. 8A) and insulin (FIG. 8B) levelspost-intraperitoneal glucose challenge in young (12-week-old), agematched male mice following an overnight fast. (FIG. 8A) plasma glucoselevels over time. (FIG. 8B) insulin clearance insulin levels,respectively, in post-intraperitoneal glucose challenge mice. N=8 pergroup. Unpaired t-test. **, p-value<0.01.

FIG. 9: Effects of a high fat diet (HFD) and low fat diet (LFD) on bodyweight of mARC2KO and WT male mice over time. mARC2 deletion inhibitsHFD induced weight gain in male mice. Male wildtype (WT, squares) andknockout (KO, circles) littermates were randomly provided either HFD(solid squares and circles) or LFD (empty squares and circles). Specialdiet was started at 9 weeks old. N=4-8. Statistical analysis (Studentst-test) was performed at each time point to compare the KO and WT miceon HFD.

FIGS. 10A-10C: Graphs showing body composition of young (12-week-old)age matched male mice. Body weight was measured before (FIG. 10A) andafter (FIG. 10B) surgical implantation of a jugular vein catheter forlater hyperinsulinemic euglycemic clamp and metabolic tracerexperiments. Fat mass (FIG. 10C, left) and lean mass (FIG. 10C, right)was also measured post-surgery, as described in Example 2, in WT (gray)and mARC2 KO (white) mice. N=8 Unpaired t-test. ***, p-value<0.001. **,p-value<0.01. *, p-value<0.05.

FIGS. 11A-11C: Graphs of plasma lipid, glucose, and insulin levels inyoung (12 week-old) male littermate mice during basal (euglycemia) andclamped (hyperinsulinemia and euglycemia) time points collected duringthe metabolic clamp experiments. Graphs demonstrate the effect of mARC2deletion on plasma fatty acids (FIG. 11A), insulin (FIG. 11B), andglucose (FIG. 11C) levels. Data collected with body-matched male12-week-old WT (gray) and mARC2 KO (white) littermate mice. N=5-6. *,p-value<0.05

FIGS. 12A-12C: Graphs of glucose concentration and glucose infusionrates (GIR) during the hyperinsulinemic euglycemic clamp experiments.Body weight matched 12-week-old male littermate mice were used. FIG. 12AGraph depicting both plasma glucose (top) and glucose infusion rates(GIR) (bottom) over time in WT (gray) and mARC2 KO (white) mice. FIG.12B is a graph showing quantification of glucose infusion rates over thelast 40 minutes of the clamp experiment (FIG. 12A) in WT (gray) andmARC2 KO (white) mice. Using mathematical manipulations, the amount ofradioactive tracer was deducted from the total glucose pool to calculatethe endogenous glucose production. (FIG. 12C).

FIGS. 13A-13E: Graphs of metabolic tracer data using radioactivelylabeled 1-¹⁴C2-deoxy glucose (2DG), demonstrating the effect of mARC2 KOon tissue specific glucose transport and metabolism. Metabolicallyactive tissues were collected: gonadal white adipose tissue (WAT) (FIG.13A), inguinal WAT (FIG. 13B), heart (FIG. 13C), brown adipose tissue(BAT) (FIG. 13D), and skeletal muscle tissue (FIG. 13E) in WT (gray) andmARC2 KO (white) mice. The glucose analogue 2DG is transported intocells and metabolized to 1-¹⁴C-2-deoxyglucose-6-phosphate (2DGP), butnot processed further (i.e., glycolysis), thus, quantification of1-¹⁴C-2-deoxyglucose-6-phosphate is a measure of intercellular glucosetransport (nmoles per gram f tissue per minute).

FIG. 14 is a graph showing the rate of glycolysis in WT (gray) and mARC2KO (white) mice. Tritiated glucose (3-³H glucose) introduced during thehyperinsulinemic euglycemic clamp was measured in plasma before andafter water evaporation to quantify the amount of radioactive glucosethat entered glycolysis. The difference in radioactivity (pre- andpost-dying) is equivalent to the amount of water removed duringevaporation.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is in no wayintended to limit the invention, its application, or uses. While thedescription is designed to permit one of ordinary skill in the art tomake and use the invention, and specific examples are provided to thatend, they should in no way be considered limiting. It will be apparentto one of ordinary skill in the art that various modifications to thefollowing will fall within the scope of the appended claims. The presentinvention should not be considered limited to the presently disclosedaspects, whether provided in the examples or elsewhere herein.

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. As used herein “a” and “an” refer to one or more.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are open ended and do not exclude the presence ofother elements not identified. In contrast, the term “consisting of” andvariations thereof is intended to be closed, and excludes additionalelements in anything but trace amounts.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings and “mammal”refers to all mammals, including, but not limited to human beings.

As used herein, the “treatment” or “treating” of obesity, hyperglycemia,diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, in a patient, means administration to a patient by anysuitable dosage regimen, procedure and/or administration route of acomposition, device, or structure with the object of achieving abeneficial or desirable clinical/medical end-point, including but notlimited to, preventing, reducing, and/or eliminating any symptom ofobesity, hyperglycemia, diabetes, metabolic syndrome, insulin resistance(insulin insensitivity), impaired glucose tolerance, high glucoselevels, pulmonary hypertension, and/or a condition arising from any ofthe foregoing, in a patient. An amount of any agent, administered by anysuitable route, effective to treat a patient is an amount capable ofpreventing, reducing, and/or eliminating any symptom of obesity,hyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, in a patient.

The compositions described herein can be administered by any effectiveroute, such as parenteral, e.g., intravenous, intramuscular,subcutaneous, intradermal, etc., formulations of which are describedbelow and in the below-referenced publications, as well as isbroadly-known to those of ordinary skill in the art.

Suitable dosage forms may include single-dose, or multiple-dose vials orother containers, such as medical syringes, containing a compositioncomprising an active ingredient useful for treatment of obesity,hyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, as described herein.

mARC2 (mitochondrial amidoxime reducing component 2, also known asMOSC2, see, GenelD: 54996, mARC2 mitochondrial amidoxime reducingcomponent 2 [Homo sapiens (human)]) is a gene encoding an enzyme foundin the outer mitochondrial membrane that reduces N-hydroxylatedsubstrates. The encoded protein uses molybdenum as a cofactor andcytochrome b5 type B and NADH cytochrome b5 reductase as accessoryproteins. One type of substrate used is N-hydroxylated nucleotide baseanalogues, which can be toxic to a cell. For example, mARC2 protectshuman cells against apoptotic effects of the base analogN⁶-hydroxylaminopurine. Other substrates includeN(omega)-hydroxy-L-arginine (NOHA) and amidoxime prodrugs, which areactivated by the encoded enzyme. Multiple transcript variants encodingthe different isoforms have been found for this gene, see, e.g.,NM_001317338.1/NP_001304267.1 (isoform a precursor, see FIGS. 1A (SEQ IDNO: 1) and 1C (SEQ ID NO: 2)), NM_001331042.1/NP_001317971.1 (isoform bprecursor), and NM_017898.4/NP_060368.2 (isoform a precursor). By mARC2,it is meant not only human mARC2, but mARC2 from any vertebrate ormammalian source, including, but not limited to, human, bovine, chicken,rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig,unless specified otherwise. The term also refers to fragments andvariants of native mARC2 that maintain at least one in vivo or in vitroactivity of a native mARC2. The term encompasses full-length unprocessedprecursor forms of mARC2, as well as mature forms resulting from furtherprocessing, e.g., from post-translational processing. In one aspect,where an iRNA agent is used to knock down expression of mARC2, thetarget portion of the sequence will be at least long enough to serve asa substrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a mARC2 gene.

mARC1 (mitochondrial amidoxime reducing component 1, Gene ID: 64757,mARC1 mitochondrial amidoxime reducing component 1 [Homo sapiens(human)], also MOSC1) also is a gene encoding an enzyme able to reduceN(omega)-hydroxy-L-arginine (NOHA) and amidoxime prodrugs, which areactivated by the encoded enzyme, see, e.g., NM_022746.3 (FIG. 1B, SEQ IDNO: 3)/NP_073583.3 (precursor) (FIG. 1D, SEQ ID NO: 4). By mARC1, it ismeant not only human mARC1, but mARC1 from any vertebrate or mammaliansource, including, but not limited to, human, bovine, chicken, rodent,mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unlessspecified otherwise. The term also refers to fragments and variants ofnative mARC1 that maintain at least one in vivo or in vitro activity ofa native mARC1. The term encompasses full-length unprocessed precursorforms of mARC1, as well as mature forms resulting from furtherprocessing, e.g., from post-translational processing. In one aspect,where an iRNA agent is used to knock down expression of mARC1, thetarget portion of the sequence will be at least long enough to serve asa substrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a mARC1 gene.

In aspects, a method of treating hyperglycemia in a patient is provided.In other aspects, a method of treating diabetes, metabolic syndrome,insulin resistance (insulin insensitivity), impaired glucose tolerance,high glucose levels, pulmonary hypertension, and/or a condition arisingfrom any of the foregoing, is provided. The patient can be human oranother animal. The hyperglycemia may relate to any number ofconditions, such as insulin resistance, metabolic syndrome, diabetes, orobesity.

Hyperglycemia is a condition in which excess glucose circulates inblood. Hyperglycemia can result, among other consequences, in kidneydamage, neurological damage, cardiovascular damage, damage to theretina, or damage to feet and legs. Diabetic neuropathy may be a resultof long-term hyperglycemia, as well as impairment of growth andsusceptibility to certain infection. Hyperglycemia may result fromdiabetes, from use of certain medications, from critical illness, suchas stroke or myocardial infarction, from stress, or from hormonalimbalances.

Diabetes is a disease that results in high blood glucose levels, due tothe body's inability to make, or to make sufficient quantities ofinsulin. Diabetes includes, for example and without limitation: Type Idiabetes; Type 2 diabetes; gestational diabetes; monogenic diabetes,e.g., neonatal diabetes mellitus or maturity onset diabetes of theyoung; and cystic fibrosis-related diabetes, as are broadly-known.Symptoms include increased thirst and urination, fatigue, and blurredvision.

Metabolic syndrome is a cluster of conditions that includes high bloodpressure, high blood sugar, excess body fat around the waist, andabnormal cholesterol or triglyceride levels, resulting in an increasedrisk of heart disease, stroke and diabetes. Symptoms may be similar todiabetes. Insulin resistance is often linked to metabolic syndrome.

Insulin resistance is the diminished ability of cells to respond to theaction of insulin in transporting glucose from the bloodstream intomuscle and other tissues. Insulin resistance often develops with obesityand is associated with prediabetes and the onset of type 2 diabetes.Insulin resistance may be defined clinically as the inability of a knownquantity of exogenous or endogenous insulin to increase glucose uptakeand utilization in an individual as much as it does in a normalpopulation. Pulmonary hypertension (e.g., pulmonary arterialhypertension) is high pulmonary artery pressure, and is measuredtypically, by right heart catheterization. Pulmonary hypertension can beassociated with insulin resistance, and as such, improvement (lessening)of insulin resistance is expected to effectively treat insulinresistance-associated pulmonary arterial hypertension. In one aspect,for example and without limitation, pulmonary arterial hypertension canbe defined, as a mean pulmonary artery pressure of ≥25 mm Hg at rest,measured during right heart catheterization.

“Obesity” or “obese” refers to being overweight, and in particulargrossly overweight. In aspects, an obese individual has a body massindex (BMI) of 30 or higher, 35 or higher, or 40 or higher.

“Expression” of a gene refers to the conversion of a DNA sequence of agene, e.g., the mARC2 gene, to an active, mature gene product such as apolypeptide/protein, or a functional nucleic acid, and includes, forexample, transcription, post-transcriptional modification (e.g.,splicing), translation, and post-translational processing and/ormodification of a protein. Expression of a gene can be reduced by anyeffective mechanism at any stage of the gene expression process, such asby affecting transcriptional activation, transcription,post-transcriptional RNA processing, translation, and post-translationalprocessing or modification. Expression of an mRNA, such as the mARC2mRNA, described herein refers to, without limitation, any aspect oftranscription of, splicing of, translation of, and post-translationalprocessing, stability, and activity of the protein product of the mRNA,e.g., the protein product of the mARC2 isoform mRNA of the mARC2 gene.Decreasing the activity of a gene product may be accomplished not onlyby decreasing expression of the active protein product, but by affectingthe mature protein product, such as by blocking, decoying, or otherwiseinterfering with the binding of the active product, or a complexcontaining the active product, to prevent its activity.

Provided herein is a method of treating obesity, hyperglycemia,diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, in a patient that comprises selectively decreasing expressionof mARC2 in a patient, e.g., in a patient's vascular tissue, adiposetissue, heart, or liver. Also provided herein is a method of treatingobesity, hyperglycemia, diabetes, metabolic syndrome, insulin resistance(insulin insensitivity), impaired glucose tolerance, high glucoselevels, pulmonary hypertension, and/or a condition arising from any ofthe foregoing, in a patient that comprises selectively decreasingexpression of mARC1 in a patient, e.g., in a patient's adipose, orvascular tissue. There are a number of ways to decrease expression oractivity of a gene in a patient, including, for example, and withoutlimitation: RNA interference, antisense technology, and inhibition ofthe activity of the mARC2 or mARC1 gene product through use of, e.g.,small molecules or agents that interfere with activity of mARC2 ormARC1, such as decoys, binding reagents, antagonists, etc. As shownherein, RNA interference (RNAi) is one method by which expression ofmARC2 or mARC1 can be specifically knocked down. Treatment of a patientresults in a decrease in one or more symptoms of obesity, hyperglycemia,diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, in a patient, such as insulin resistance or high glucoselevels.

Drug products, or pharmaceutical compositions comprising an active agent(e.g., drug), for example, an active agent that decreases mARC2expression or activity, or mARC1 expression or activity may be preparedby any method known in the art of pharmacy, for example, by bringinginto association the active ingredient with the carrier(s) orexcipient(s). As used herein, a “pharmaceutically acceptable excipient”,“carrier” or “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptableexcipients include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it may be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the activeagent. In certain aspects, the active compound may be prepared with acarrier that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used in delivery systems, such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid. Many methods for the preparation of suchformulations are broadly-known to those skilled in the art.

Additionally, active agent-containing compositions may be in variety offorms. The preferred form depends on the intended mode of administrationand therapeutic application, which will in turn dictate the types ofcarriers/excipients. Suitable forms include, but are not limited to,liquid, semi-solid and solid dosage forms.

Pharmaceutical formulations adapted for oral administration may bepresented, for example and without limitation, as discrete units such ascapsules or tablets; powders or granules; solutions or suspensions inaqueous or non-aqueous liquids; edible foams or whips; or oil-in-waterliquid emulsions or water-in-oil liquid emulsions. In certainembodiments, the active agent may be contained in a formulation suchthat it is suitable for oral administration, for example, by combiningthe active agent with an inert diluent or an assimilable edible carrier.The active agent (and other ingredients, if desired) may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Pharmaceutical formulations adapted for transdermal administration maybe presented, for example and without limitation, as discrete patchesintended to remain in intimate contact with the epidermis of therecipient for a prolonged period of time or electrodes for iontophoreticdelivery.

Pharmaceutical formulations adapted for topical administration may beformulated, for example and without limitation, as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays,aerosols, or oils.

Pharmaceutical formulations adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size, forexample, in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalationinclude, without limitation, fine particle dusts or mists which may begenerated by means of various types of metered dose pressurizedaerosols, nebulizers, or insufflators. In the context of delivery of theactive agents described herein by inhalation, inhalation drug products,such as metered-dose inhalers, as are broadly-known in thepharmaceutical arts, are used. Metered dose inhalers are configured todeliver a single dose of an active agent per actuation, though multipleactuations may be needed to effectively treat a given patient.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain, for example and without limitation, anti-oxidants, buffers,bacteriostats, lipids, liposomes, emulsifiers, also suspending agentsand rheology modifiers. The formulations may be presented in unit-doseor multi-dose containers, for example, sealed ampoules and vials, andmay be stored in a freeze-dried (lyophilized) condition requiring onlythe addition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. For example, sterile injectablesolutions can be prepared by incorporating the active agent in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, typical methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

A “therapeutically effective amount” refers to an amount of a drugproduct or active agent effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. An “amounteffective” for treatment of a condition is an amount of an active agentor dosage form, such as a single dose or multiple doses, effective toachieve a determinable end-point. The “amount effective” is preferablysafe—at least to the extent the benefits of treatment outweighs thedetriments, and/or the detriments are acceptable to one of ordinaryskill and/or to an appropriate regulatory agency, such as the U.S. Foodand Drug Administration. A therapeutically effective amount of an activeagent may vary according to factors such as the disease state, age, sex,and weight of the individual, and the ability of the active agent toelicit a desired response in the individual. A “prophylacticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired prophylactic result.Typically, since a prophylactic dose is used in subjects prior to or atan earlier stage of disease, the prophylactically effective amount maybe less than the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time, or the composition may be administered continuously or in apulsed fashion with doses or partial doses being administered at regularintervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120minutes, every 2 through 12 hours daily, or every other day, etc., beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. In some instances, it may be especiallyadvantageous to formulate compositions, such as parenteral or inhaledcompositions, in dosage unit form for ease of administration anduniformity of dosage. The specification for the dosage unit forms aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic or prophylacticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

By “target-specific” or reference to the ability of one compound to bindanother target compound specifically, it is meant that the compoundbinds to the target compound to the exclusion of others in a givenreaction system, e.g., in vitro, or in vivo, to acceptable tolerances,permitting a sufficiently specific diagnostic or therapeutic effectaccording to the standards of a person of skill in the art, a medicalcommunity, and/or a regulatory authority, such as the U.S. Food and DrugAgency (FDA), in aspects, in the context of targeting mARC2, anddown-regulating mARC2 activity, or targeting mARC1, and down-regulatingmARC1 activity, and effectively treating obesity, hyperglycemia,diabetes, metabolic syndrome, insulin resistance (insulininsensitivity), impaired glucose tolerance, high glucose levels,pulmonary hypertension, and/or a condition arising from any of theforegoing, in a patient, as described herein.

A “binding reagent” is a reagent, compound or composition, e.g., aligand, able to specifically bind a target compound, such as mARC2 ormARC1. A binding reagent can interfere with mARC2 or mARC1 activity, forexample as an antagonist or decoy within the context of mARC2's activityor mARC1's activity. Binding reagents include, without limitation,antibodies (polyclonal, monoclonal, humanized, etc.), antibody fragments(e.g., a recombinant scFv), antibody mimetics such as affibodies,affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins,fynomers, monobodies, nucleic acid ligands (e.g., aptamers), engineeredproteins, antigens, epitopes, haptens, or any target-specific bindingreagent. In aspects, binding reagents includes as a class: monoclonalantibodies, or derivatives or analogs thereof, including withoutlimitation: Fv fragments, single chain Fv (scFv) fragments, Fab′fragments, F(ab′)₂ fragments, single domain antibodies, camelizedantibodies and antibody fragments, humanized antibodies and antibodyfragments, multivalent versions of the foregoing, and anyparatope-containing compound or composition; multivalent activatorsincluding without limitation: monospecific or bispecific antibodies,such as disulfide stabilized Fv fragments, scFv tandems ((scFv)₂fragments), diabodies, tribodies or tetrabodies, which typically arecovalently linked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; nucleic acids and analogs thereof that binda target compound; or receptor molecules which naturally interact with adesired target molecule. Anti-mARC2 antibodies and anti-mARC1 antibodiesare commercially-available, or can be generated by a person of ordinaryskill in the art using common methods.

A “gene” is a sequence of DNA or RNA which codes for a molecule, such asa protein or a functional RNA that has a function. Nucleic acids arebiopolymers, or small biomolecules, essential to all known forms oflife. They are composed of nucleotides, which are monomers made of threecomponents: a 5-carbon sugar, a phosphate group and a nitrogenous base.If the sugar is a simple ribose, the polymer is RNA; if the sugar isderived from deoxyribose, the polymer is DNA. DNA typically uses thenitrogenous bases guanine, thymine, adenine, and cytosine. RNA typicallyuses the nitrogenous bases guanine, uracil, adenine, and cytosine.

Complementary refers to the ability of polynucleotides (nucleic acids)to hybridize to one another, forming inter-strand base pairs. Base pairsare formed by hydrogen bonding between nucleotide units in antiparallelpolynucleotide strands. Complementary polynucleotide strands can basepair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C toG), or in any other manner that allows for the formation of duplexes.When using RNA as opposed to DNA, uracil rather than thymine is the basethat is considered to be complementary to adenosine. Two sequencescomprising complementary sequences can hybridize if they form duplexesunder specified conditions, such as in water, saline ((e.g., normalsaline, or 0.9% w/v saline) or phosphate-buffered saline), or underother stringency conditions, such as, for example and withoutlimitation, 0.1×SSC (saline sodium citrate) to 10×SSC, where 1×SSC is0.15M NaCl and 0.015M sodium citrate in water. Hybridization ofcomplementary sequences is dictated, e.g., by salt concentration andtemperature, with the melting temperature (Tm) lowering with increasedmismatches and increased stringency. Perfectly matched sequences aresaid to be fully complementary, or have 100% sequence identity (gaps arenot counted and the measurement is in relation to the shorter of the twosequences). In one aspect, a sequence that “specifically hybridizes” toanother sequence, does so in a hybridization solution containing 0.5Msodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA, and 100mg/ml of salmon sperm DNA at 65° C. for 16 hours and washing twice at65° C. for twenty minutes in a washing solution containing 0.5×SSC and0.1% SDS, or does so under conditions more stringent than 2×SSC at 65°C., for example, in 0.2×SSC at 55° C. A sequence that specificallyhybridizes to another typically has at least 80%, 85%, 90%, 95%, or 99%sequence identity with the other sequence.

Gene expression is the process by which information from a gene is usedin the synthesis of a functional gene product, e.g., a protein orfunctional RNA. Gene expression involves various steps, includingtranscription, translation, and post-translational modification of aprotein.

Transcription is the process by which the DNA gene sequence istranscribed into pre-mRNA (messenger RNA). The steps include: RNApolymerase, together with one or more general transcription factors,binds to promoter DNA. Transcription factors (TFs) are proteins thatcontrol the rate of transcription of genetic information from DNA tomessenger RNA, by binding to a specific DNA sequence (i.e., the promoterregion). The function of TFs is to regulate genes in order to make surethat they are expressed in the right cell at the right time and in theright amount throughout the life of the cell and the organism. Thepromoter region of a gene is a region of DNA that initiatestranscription of that particular gene. Promoters are located near thetranscription start sites of genes, on the same strand, and often, butnot exclusively, are upstream (towards the 5′ region of the sensestrand) on the DNA. Promoters can be about 100-1000 base pairs long.Additional sequences and non-coding elements can affect transcriptionrates. If the cell has a nucleus (eukaryotes), the RNA is furtherprocessed. This includes polyadenylation, capping, and splicing.Polyadenylation is the addition of a poly(A) tail to a messenger RNA.The poly(A) tail consists of multiple adenosine monophosphates; in otherwords, it is a stretch of RNA that has only adenine bases. Ineukaryotes, polyadenylation is part of the process that produces maturemessenger RNA (mRNA) for translation. Capping refers to the processwherein the 5′ end of the pre-mRNA has a specially altered nucleotide.In eukaryotes, the 5′ cap (cap-0), found on the 5′ end of an mRNAmolecule, consists of a guanine nucleotide connected to mRNA via anunusual 5′ to 5′ triphosphate linkage. During RNA splicing, pre-mRNA isedited. Specifically, during this process introns are removed and exonsare joined together. The resultant product is known as mature mRNA. TheRNA may remain in the nucleus or exit to the cytoplasm through thenuclear pore complex.

RNA levels in a cell, e.g., mRNA levels, can be controlledpost-transcriptionally. Native mechanisms, including: endogenous genesilencing mechanisms, interference with translational mechanisms,interference with RNA splicing mechanisms, and destruction of duplexedRNA by RNAse H, or RNAse H-like, activity. As is broadly-recognized bythose of ordinary skill in the art, these endogenous mechanisms can beexploited to decrease or silence mRNA activity in a cell or organism ina sequence-specific, targeted manner. Antisense technology typicallyinvolves administration of a single-stranded antisense oligonucleotide(ASO) that is chemically-modified, e.g., as described herein, forbio-stability, and is administered in sufficient amounts to effectivelypenetrate the cell and bind in sufficient quantities to target mRNAs incells. RNA interference (RNAi) harnesses an endogenous and catalyticgene silencing mechanism, which means that once, e.g., a microRNA, ordouble-stranded siRNA has been delivered, either by conjugation or innanoparticles into the cytosol, they are efficiently recognized andstably incorporated into the RNA-induced silencing complex (RiSC) toachieve prolonged gene silencing. Both antisense technologies and RNAihave their strengths and weaknesses, either may be used effectively todecrease or silence expression of a gene or gene product, such as mARC2or mARC1 (see, e.g., Watts, J. K., et al. Gene silencing by siRNAs andantisense oligonucleotides in the laboratory and the clinic (2012)226(2):365-379).

The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interferenceagent” as used interchangeably herein, refer to an agent that containsRNA as that term is defined herein, and which mediates the targetedcleavage of an RNA transcript via an RNA-induced silencing complex(RISC) pathway. iRNA directs the sequence-specific degradation of mRNAthrough a process known as RNA interference (RNAi). The iRNA modulates,e.g., inhibits or knocks down, the expression of mARC2 or mARC1 mRNA ina cell, e.g., a cell within a subject, such as a mammalian subject.

In one aspect, an RNAi agent includes a single stranded RNAi thatinteracts with a target RNA sequence, e.g., a mARC2 or mARC1 mRNAsequence, to direct the cleavage of the target RNA. Without wishing tobe bound by theory it is believed that long double stranded RNAintroduced into cells is broken down into double stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer. Dicer, aribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs. ThesesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition. Upon bindingto the appropriate target mRNA, one or more endonucleases within theRISC cleave the target to induce silencing. Thus, in one aspect theinvention relates to a single stranded RNA (ssRNA) (the antisense strandof an siRNA duplex) generated within a cell and which promotes theformation of a RISC complex to effect silencing of the target gene.Accordingly, the term “siRNA” is also used herein to refer to aninterfering RNA (iRNA).

In another aspect, the RNAi agent may be a single-stranded RNA that isintroduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No.8,101,348, incorporated herein by reference for its technicaldisclosure, and in Lima et al., (2012) Cell 150:883-894. Any of the RNAiagents described herein may be used as a single-stranded siRNA asdescribed herein or as chemically modified by the methods described inLima et al.

In another aspect, an “iRNA” or iRNA agent” for use in the compositionsand methods described herein is a double stranded RNA and can bereferred to herein as a “double stranded RNAi agent,” “double strandedRNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”,refers to a complex of ribonucleic acid molecules, having a duplexstructure comprising two anti-parallel and substantially complementarynucleic acid strands, referred to as having “sense” and “antisense”orientations with respect to a target RNA, i.e., a mARC2 mRNA or a mARC1mRNA. In some aspects, a double stranded RNA (dsRNA) triggers thedegradation of a target RNA, e.g., an mRNA, through apost-transcriptional gene-silencing mechanism referred to herein as RNAinterference or RNAi.

The majority of nucleotides of each strand of a dsRNA molecule may beribonucleotides, but as described in detail herein, each or both strandscan also include nucleotide analogs, where one or morenon-ribonucleotides, e.g., a deoxyribonucleotide and/or a modifiednucleotide. In addition, as used in this specification, an “RNAi agent”or “RNAi reagent” may include ribonucleotides with chemicalmodifications; an RNAi agent may include substantial modifications atmultiple nucleotides. As used herein, the term “modified nucleotide”refers to a nucleotide having, independently, a modified sugar moiety, amodified inter-nucleotide linkage, and/or modified nucleobase. Thus, theterm modified nucleotide encompasses substitutions, additions or removalof, e.g., a functional group or atom, to inter-nucleoside linkages,sugar moieties, or nucleobases. The modifications suitable for use inthe reagents described herein include all types of modificationsdisclosed herein or known in the art. Any such modifications, as used ina siRNA type molecule, are encompassed by “RNAi agent” or “RNAi reagent”for the purposes of this disclosure.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome aspects, the hairpin loop can comprise at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 20, at least 23, or more unpaired nucleotides. Insome aspects, the hairpin loop can be 10 or fewer nucleotides. In someaspects, the hairpin loop can be 8 or fewer unpaired nucleotides. Insome aspects, the hairpin loop can be 4-10 unpaired nucleotides. In someaspects, the hairpin loop can be 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In one aspect, an RNAi agent is a dsRNA, each strand of which comprises19-23 nucleotides, that interacts with a target RNA sequence, e.g., amARC2 mRNA or a mARC1 mRNA, without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer. Dicer, a ribonuclease-III-likeenzyme, processes the dsRNA into 19-23 base pair short interfering RNAswith characteristic two base 3′ overhangs. The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition. Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing. In one aspect, an RNAi agent is adsRNA of 24-30 nucleotides that interacts with a target RNA sequence,e.g., a mARC2 or mARC1 target mRNA sequence, to direct the cleavage ofthe target RNA.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one aspect of the dsRNA, at least one strand comprises a 3 ‘ overhangof at least 1 nucleotide. In another aspect, at least one strandcomprises a 3’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other aspects, at leastone strand of the RNAi agent comprises a 5′ overhang of at least 1nucleotide. In certain aspects, at least one strand comprises a 5′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In still other aspects, both the 3′ andthe 5′ end of one strand of the RNAi agent comprise an overhang of atleast 1 nucleotide.

In one aspect, the antisense strand of a dsRNA has a 1-10 nucleotide,e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the3′-end and/or the 5′-end. In one aspect, the sense strand of a dsRNA hasa 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In certain aspects, theoverhang on the sense strand or the antisense strand, or both, caninclude extended lengths longer than 10 nucleotides, e.g., 1-30nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotidesin length. In certain aspects, an extended overhang is on the sensestrand of the duplex. In certain aspects, an extended overhang ispresent on the 3′end of the sense strand of the duplex. In certainaspects, an extended overhang is present on the 5′end of the sensestrand of the duplex. In certain aspects, an extended overhang is on theantisense strand of the duplex. In certain aspects, an extended overhangis present on the 3′end of the antisense strand of the duplex. Incertain aspects, an extended overhang is present on the 5′end of theantisense strand of the duplex. In another aspect, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt.

Where both ends of a dsRNA are blunt, the dsRNA is said to be bluntended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt atboth ends, i.e., no nucleotide overhang at either end of the molecule.Most often such a molecule will be double stranded over its entirelength.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a mARC2 mRNA or a mARC1 mRNA.As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example, a target sequence, e.g., a mARC2 mRNA sequence ora mARC1 mRNA sequence, e.g., as described herein. Where the region ofcomplementarity is not fully complementary to the target sequence, themismatches can be in the internal or terminal regions of the molecule.Generally, the most tolerated mismatches are in the terminal regions,e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus ofthe IRNA.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some aspects, thecleavage region comprises three bases on either end of, and immediatelyadjacent to, the cleavage site. In some aspects, the cleavage regioncomprises two bases on either end of, and immediately adjacent to, thecleavage site. In some aspects, the cleavage site specifically occurs atthe site bound by nucleotides 10 and 11 of the antisense strand, and thecleavage region comprises nucleotides 11, 12 and 13.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an IRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of a messenger RNA (mRNA)” refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., a mARC2 mRNA).

Accordingly, in some aspects, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target mARC2 mRNAsequence. In other aspects, the antisense strand polynucleotidesdisclosed herein are substantially complementary to the target mARC2mRNA sequence and comprise a contiguous nucleotide sequence which is atleast about 80% complementary over its entire length to the equivalentregion of the nucleotide sequence of SEQ ID NO: 1, or a fragmentthereof, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, or about 99% complementary.

It is understood that the sequence of the mARC2 mRNA or the sequence ofthe mARC1 mRNA must be sufficiently complementary to the antisensestrand of the iRNA agent for the agent to be used in the indicatedpatient, e.g. human, mammalian, or vertebrate species.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing”, “knockingdown”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a mARC2 isoform mRNA,” as usedherein, includes inhibition of expression of any mARC2 gene (such as,e.g., a mouse mARC2 gene, a rat mARC2 gene, a monkey mARC2 gene, or ahuman mARC2 gene) as well as variants or mutants of an mARC2 gene thatencode a mARC2 protein, in its production of mARC2 mRNA, affecting thestability of mARC2 mRNA, such as by antisense or RNAi technologies, orinhibiting translation of mARC2 mRNA. The phrase “inhibiting expressionof a mARC1 isoform mRNA,” as used herein, includes inhibition ofexpression of any mARC1 gene (such as, e.g., a mouse mARC1 gene, a ratmARC1 gene, a monkey mARC1 gene, or a human mARC1 gene) as well asvariants or mutants of an mARC1 gene that encode a mARC1 protein, in itsproduction of mARC1 mRNA, affecting the stability of mARC1 mRNA, such asby antisense or RNAi technologies, or inhibiting translation of mARC1mRNA.

“Inhibiting expression of a mARC2 mRNA” includes any level of inhibitionof a mARC2 mRNA, e.g., at least partial suppression of the expression ofa mARC2 mRNA, such as an inhibition by at least about 20%. “Inhibitingexpression of a mARC1 mRNA” includes any level of inhibition of a mARC1mRNA, e.g., at least partial suppression of the expression of a mARC1mRNA, such as an inhibition by at least about 20%. In certain aspects,inhibition is by at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%.

The expression of a mARC2 mRNA may be assessed based on the level of anyvariable associated with mARC2 mRNA expression, e.g., mARC2 mRNA levelor mARC2 protein level. The expression of a mARC2 mRNA may also beassessed indirectly based on assay of physiological markers associatedwith decreased expression of the mARC2 mRNA in a patient, such as bloodglucose levels. Likewise, the expression of a mARC1 mRNA may be assessedbased on the level of any variable associated with mARC1 mRNAexpression, e.g., mARC1 mRNA level or mARC1 protein level. Theexpression of a mARC1 mRNA may also be assessed indirectly based onassay of physiological markers associated with decreased expression ofthe mARC1 mRNA in a patient, such as blood glucose levels.

In one aspect, at least partial suppression of the expression of a mARC2mRNA, or of the expression of a mARC1 mRNA, is assessed by a reductionof the amount of mARC2 mRNA or mARC1 mRNA, respectively, which can beisolated from or detected in a first cell or group of cells, e.g., mARC2is expressed in the following (in order of highest to lowest expression)liver (hepatocytes); Thyroid, adipose tissues: white (gonadal), beige(inguinal), and brown; vascular tissue (endothelial, smooth muscle,fibroblasts)—mainly pulmonary vasculature; heart (cardiomyocytes), lung(type 2 endothelial cells and epithelial cells), and immune cells(macrophages). mARC1 is abundant in the liver (hepatocytes); adiposetissue (white (gonadal), beige (inguinal), and brown, gonadal; vasculartissue (endothelial, smooth muscle). As such, in aspects, mARC2 and/ormARC1 levels are determined in liver, in adipose tissue, or in vasculartissue (e.g., heart and arteries for mARC2). A reduction of the amountof mARC2 mRNA or mARC1 mRNA, respectively, in a cell or tissue in whicha mARC2 gene or a mARC1 gene, respectively, is transcribed and which hasbeen treated such that the expression of a mARC2 mRNA, or of a mARC1mRNA, is inhibited, is determined as compared to a second cell or tissuesubstantially identical to the first cell or tissue but which has notbeen so treated (control cells). The degree of inhibition may beexpressed in terms of:

$\left. {\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \times 100\%} \right)$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the iRNA or contacting a cell in vivo with the iRNA. The contactingmay be done directly or indirectly. Thus, for example, the RNAi agentmay be put into physical contact with the cell by the individualperforming the method, or alternatively, the RNAi agent may be put intoa situation that will permit or cause it to subsequently come intocontact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GaINAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

In one aspect, contacting a cell with an iRNA includes “introducing” or“delivering the iRNA into the cell” by facilitating or effecting uptakeor absorption into the cell. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. Introducing an iRNA into a cell may be in vitroand/or in vivo. For example, for in vivo introduction, iRNA can beinjected into a tissue site or administered systemically. In vivodelivery can also be done by a beta-glucan delivery system, such asthose described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.Patent Application Publication No. 2005/0281781, the technicaldisclosure of which are hereby incorporated herein by reference. Invitro introduction into a cell includes methods known in the art such aselectroporation and lipofection. Further approaches are described hereinbelow and/or are known in the art.

As used herein, and further to the discussion above regarding iRNAreagents, “agent” or “i RNA agent”, when used in the context of anantisense, RNAi, or ribozyme, or other single-stranded ordouble-stranded RNA interfering nucleic acids, refers not only to RNAstructures, but effective nucleic acid analog structures. In antisenseand RNAi technologies, use of RNA poses significant delivery issues dueto the lability of RNA molecules. As such, RNA is commonlychemically-modified to produce nucleic acid analogs, not only to enhancestability of the nucleic acid molecules, but often resulting inincreased binding affinity, and with reduced toxicity. Suchmodifications are broadly-known to those of ordinary skill in the art,and are available commercially (see, e.g., Corey, D. R., Chemicalmodification: the key to clinical application of RNA interference?(2007) J Clin Invest. 117(12):3615-3622, also describing RNAi, andUnited States Patent Application Publication No. 2017/0081667,incorporated herein by reference for its techical disclosure).Non-limiting examples of modifications to the nucleic acid structure innucleic acid analogs include: modifications to the phosphate linkage,such as phosphoramidates or phosphorothioates; sugar modification, suchas 2′-O, 4′-C methylene bridged, locked nucleic acid (LNA), 2′-methoxy,2′-O-methoxyethyl (MOE), 2′-fluoro, S-constrained-ethyl (cEt), andtricyclo-DNA (tc-DNA); and non-ribose structures, such asphosphorodiamidate morpholino (PMO) and peptide-nucleic acids (PNA).

In addition to those mARC2-active IRNA agents, and mARC1-active IRNAagents, described herein, antisense reagents (ASOs), other RNAi agents,ribozyme reagents, and other nucleic acid-based methods of reducing geneexpression, can be designed and tested based on known sequences of mARC2or mARC1 RNAs and gene structure (exemplary sequences are providedherein and the mARC2 and mARC1 genes are well-studied). Based on thepresent disclosure, one of ordinary skill can design, and/or produce anactive agent capable of knocking down mARC2 or mARC1 expression. Ofnote, a number of publications describe algorithms for generatingcandidate iRNA sequences, and publically-available software can be usedto implement those algorithms. As such, typically, one only needs toenter an mRNA sequence into a calculator to produce candidate iRNAs.

Exemplary locations for iRNA specific to mARC2 mRNA include, withoutlimitation, and in reference to the sequence of NM_001317338.1, shown inFIG. 1A: 479, 690, 784, 789, 1004, 1106, 1041, 1073, 1106, 1128, 1417,and are commercially available from Dharmacon of Lafayette, Colo., andThermoFisher Scientific, among others. In one aspect, the siRNAspecifically hybridizes to a nucleic acid having the sequence of SEQ IDNO: 1, or a sequence complementary thereto. In another aspect, the siRNAspecifically hybridizes to an mRNA sequence encoding a mARC2 protein orthe protein of SEQ ID NO 2, or a sequence complementary thereto. Inanother aspect, the siRNA specifically hybridizes to a nucleic acidhaving the sequence of bases 559-1332 of SEQ ID NO: 1, or a sequencecomplementary thereto. Table A provides sequences of exemplary siRNAreagents.

TABLE A Exemplary RNAi agents (siRNA) for knockingdown expression of mARC2 in humans Sequence SEQ ID Location (5′ → 3′)NO.  620 GTGCTCATCTCCATCATTTAT  5 1046 GATGAACTCCTAATTGGTAGT  6 1049GAACTCCTAATTGGTAGTGTA  7 1347 GAGGGATTGACTGAGATCTTA  8 1368ACAACAGCAGCAACGATACAT  9 1751 GATGTGCAGGACGCATGTTAC 10

Exemplary locations for iRNA specific to mARC1 mRNA include, withoutlimitation, and in reference to the sequence of NM_022746.3, shown inFIG. 1B: 539, 641, 652, 656, 819, 858, 936, 969, 970, 1009, 1697, and2068, and are commercially available from Dharmacon of Lafayette, Colo.,and ThermoFisher Scientific, among others. In one aspect, the siRNAspecifically hybridizes to a nucleic acid having the sequence of SEQ IDNO: 3, or a sequence complementary thereto. In another aspect, the siRNAspecifically hybridizes to an mRNA sequence encoding a mARC1 protein orthe protein of SEQ ID NO 4, or a sequence complementary thereto. Inanother aspect, the siRNA specifically hybridizes to a nucleic acidhaving the sequence of bases 309-1135 of SEQ ID NO: 3, or a sequencecomplementary thereto. Table B provides sequences of exemplary siRNAreagents.

TABLE B Exemplary RNAi agents (siRNA) for knockingdown expression of mARC1 in humans Sequence SEQ ID Location (5′ → 3′)NO.  644 GGACCTACTACTGCCTATCAA 11 1130 GAAGAGTTATCGCCAGTGTGA 12 1149GACCCTTCAGAACGAAAGTTA 13 1407 GGTGTCTCAATGCTTCAATGT 14 1645GGCTGGAAGAATATCCTAGAA 15 2018 GTGATTTCAGATAGACTACTG 16

Therefore, according to one aspect, provided herein is a method oftreating obesity, hyperglycemia, diabetes, metabolic syndrome, insulinresistance (insulin insensitivity), impaired glucose tolerance, highglucose levels, pulmonary hypertension, and/or a condition arising fromany of the foregoing, in a patient, comprising reducing mARC2 or mARC1expression or activity to a level effective to treat one or moresymptoms of obesity, hyperglycemia, diabetes, metabolic syndrome,insulin resistance (insulin insensitivity), impaired glucose tolerance,high glucose levels, pulmonary hypertension, and/or a condition arisingfrom any of the foregoing, in a patient. By “reducing activity” of agene or gene product, e.g., a mARC2 mRNA, or a mARC1 mRNA, it is meant,by any method decreasing, suppressing, or silencing expression of thegene, decreasing activity of the gene product, and/or reducing availablelevels of the gene product in the patient. Activity of a mARC2 mRNA, ora mARC1 mRNA, can be reduced, e.g., by use of antisense nucleic acids,or by use of iRNA agents. Activity of a mARC2 mRNA, or of a mARC1 mRNA,also can be reduced, e.g., by antagonism, or otherwise blocking orinterfering with the activity of a mARC2 mRNA, or mARC1 mRNA,respectively, or by mutation. Available levels of the gene product canbe reduced in a patient, for example, either systemically or locally,for example in a patient's adipose, vascular, or hepatic tissue, e.g.,by binding of a mARC2 mRNA or gene product with a mARC2 binding reagent,such as an antibody, an antibody fragment, or an anti-mARC2paratope-containing polypeptide compositions, or a decoy comprising amARC2 substrate, or in the case of mARC1, by binding of a mARC1 mRNA orgene product with a mARC1 binding reagent, such as an antibody, anantibody fragment, or an anti-mARC1 paratope-containing polypeptidecompositions, or a decoy comprising a mARC1 substrate.

In aspects, by decreasing, down-regulating, or knocking down mARC2 mRNAexpression or activity, it is meant any action that results in loweractivity of mARC2 in a cell or patient—typically by use of a therapeuticagent. In one aspect, it refers to reducing the amount of mARC2 mRNAavailable for translation. In aspects, by decreasing, down-regulating,or knocking down mARC1 mRNA expression or activity, it is meant anyaction that results in lower activity of mARC1 in a cell orpatient—typically by use of a therapeutic agent. In one aspect, itrefers to reducing the amount of mARC1 mRNA available for translation.Useful therapeutic agents include, without limitation, antisense or RNAicompositions; binding reagents, such as antibodies (including antibodyfragments or antibody-based polypeptide ligands), and aptamers;antagonists; decoys; and peptide-based therapies.

U.S. Pat. No. 7,737,265 and International Patent Publication No. WO2016/209862, each of which is incorporated herein by reference for itstechnical disclosure to the extent it is consistent with the presentdisclosure, are examples of the many publications disclosing furtherdetails regarding iRNA technology and reagents, the disclosure of whichis broadly applicable to methods of making and using reagents for use inknocking down mARC2 expression or mARC1 expression, as described herein.Disclosed in WO/2016/209862 are details relating to iRNA structure,definition of required sequences and reagent size, definitions anddescriptions of target sequences, methods of making iRNAs, variations ormodifications in iRNA structures, such as nucleic acid analogs ormimetics, methods of modification of iRNAs such as ligand-modifiediRNAs, including polysaccharide-modified or polypeptide-modified iRNAsand linkers that can be useful in targeting the iRNA, pharmaceuticalcompositions for delivery of iRNAs, delivery methods and delivery routesfor iRNAs, including liposome or micellar delivery systems, and methodsof determining whether iRNAs are effective. One of ordinary skill canidentify and optimize mARC2 RNAi agents based on available knowledge andresources. Further disclosure of how to identify, make, or use mARC2RNAi reagents is unnecessary. Likewise, one of ordinary skill canidentify and optimize mARC1 RNAi agents based on available knowledge andresources. Further disclosure of how to identify, make, or use mARC1RNAi reagents is unnecessary.

In aspects, a method of treating hyperglycemia in a patient is provided.The patient can be human or another animal. The hyperglycemia may relateto any number of conditions, such as insulin resistance, metabolicsyndrome, diabetes, or obesity. As indicated herein, inhibition ofexpression or activity of mARC2 is able to reduce fat accumulation inadipose tissue, and improve glucose homeostasis and decrease insulinresistance, by, e.g. reducing both plasma glucose levels and insulinlevels during glucose tolerance tests. The method comprises decreasingmARC2 expression or activity in the patient, such that plasma glucoselevels are lowered in the patient or insulin resistance is reduced inthe patient. By virtue of lowering plasma glucose levels or insulinresistance in the patient, conditions such as chronic or acutehyperglycemia, metabolic syndrome, or diabetes, such as Type 1 Diabetes,or Type 2 Diabetes are treated. In one aspect expression of the mARC2gene is silenced by administration of an RNAi agent to the patient, suchas a siRNA, as described above and which are commercially available.

In another aspect, expression or activity of mARC1 is expected to beable to reduce fat accumulation in adipose tissue, and improve glucosehomeostasis and decrease insulin resistance, by, e.g. reducing bothplasma glucose levels and insulin levels during glucose tolerance tests.The method comprises decreasing mARC1 expression or activity in thepatient, such that plasma glucose levels are lowered in the patient orinsulin resistance is reduced in the patient. By virtue of loweringplasma glucose levels or insulin resistance in the patient, conditionssuch as chronic or acute hyperglycemia, metabolic syndrome, or diabetes,such as Type 1 Diabetes, or Type 2 Diabetes are treated. In one aspectexpression of the mARC1 gene is silenced by administration of an RNAiagent to the patient, such as a siRNA, as described above and which arecommercially available.

Example 1—Targeting of mARC2 for Improving Glucose Clearance and InsulinSensitivity

Improving insulin action has implications in several metabolic diseases(for example and without limitation, pulmonary hypertension, type 2diabetes, and metabolic syndrome). Deleting mARC2 in mice causesimproved insulin action. This leads to improved glucose utilization,decreased body fat, decreased free fatty acids, and improved energyexpenditure.

Phenotyping data from the International Mouse Phenotyping Consortium(IMPC) reports a body composition & metabolism phenotype. Significantdifferences in body composition are noted. Increased lean mass anddecreased fat mass in 3 month old male and female mice.

mARC1 and mARC2 levels change in response to nutritional status. Theyare upregulated at transcript and protein levels in liver cells exposedto elevated glucose. They are downregulated after fasting in the liversof humans, rats and mice. They are mARC2 protein expression isupregulated in mice fed a high fat diet. No change is seen in mARC2 ormARC1 protein expression in obese mice. Lastly, we found no change inmARC2 or mARC 1 transcript levels in liver of C57BI6 mice or AKR strainin response to 22 weeks of high fat diet, relative to low fat diet(LFD). Recent studies have suggested a role for mARC2 and mARC1 inlipogenesis or lipid synthases. These studies have measured differencesin lipid levels in differentiated adipocytes in which mARC2 was deletedusing siRNA.

We have completed the following studies to investigate the role of mARC2in lipogenesis and explore the physiological function of the mARCproteins. Biochemistry data demonstrates that human mARC1 and mARC-2 cangenerate nitric oxide from nitrite. We also tested the effect of mARC2on lipid uptake in primary hepatocytes isolated from mARC2 knockoutmice. Knocking down mARC2 expression in adipocytes lead to decreasedlipid accumulation via Nile red assay. We repeated this using primaryhepatocytes with and without mARC2 and measured no difference in lipidlevels.

Materials and Methods

mARC2 KO mice: The C57BL/6N mARC^(tm2A) (mARC2 KO) mouse embryos wereobtained from Knock-Out Mouse Project (KOMP) repository, thenrederivatized by Jackson laboratories. Standard breeding and husbandrymethods were used to establish and maintain a colony of mARC2 KO mice.Male mice used for experiments were generated by heterozygote breeding.Deletion of mARC2 was confirmed in multiple organs by PCR and westernblot.

Metabolic phenotyping: Mice were housed in metabolic cages to measureparameters of whole-body energy balance, measures of energy expenditureby indirect calorimetry, activity, and feeding and drinking behavior.

Diet induced obesity: We plan to conduct a comprehensive evaluation ofmetabolic function during basal (LFD; Research Diets, D12450J: 10% fat,70% carbohydrate, 20% protein) and nutritionally stressed (HFD; ResearchDiets, D12492: 60% fat, 20% carbohydrate, 20% protein) conditions. At 8weeks of age, 20 male WT and mARC2^(Frt/Floxed) KO mice ill be randomlyassigned to either a LFD or HFD, so that final group sizes will be 10mice per genotype, per diet. Body weight and composition (fat and leanmass by proton-NMR) will we be measured at baseline, and then mice willbe maintained on diets for 12 weeks. During dietary challenge, bodyweight and feeding

Metabolic clamp experiments: Hyperinsulinemic Euglycemic Clampsexperiments were performed in conjunction with Metabolic tracer studiesbelow and according the Mouse Metabolic Phenotyping Center Consortium.An indwelling catheter was surgically implanted in the right jugularvein 1 week prior to clamp experiments. Prior to experiments, mice werefasted for 6 hours. First, to measure basal glucose turnover, mice wereinfused with 3-³H-glucose at a rate of 0.05 μCi•min⁻¹ for 120 min in the“basal” euglycemia phase. Next, during the “clamp” phase, mice wereinfused with a constant rate of labeled 3-³H glucose (0.1 μCi•min⁻¹) andinsulin (2.5 mU•kg⁻¹ lean mass•min⁻¹) to induce hyperinsulinemia inaddition to euglycemia. The rate of 20% dextrose infusion was thenadjusted dynamically to maintain similar levels of euglycemia in the WTand mARC2 KO in each mouse over 120 min. The flow rate, measured by aflow meter attached to the dextrose solution, was recorded and isreported as glucose infusion rate (GIR). Blood was collected via tailevery ten minutes to monitor glucose concentration in real time with ahand-held glucose oxidase monitor. Additionally, blood was collected atthe 0 min (Basal) and 120 min (Clamp) time points for later analysis ofplasma glucose, insulin, fatty acid, and tracer levels. Glucose,insulin, and fatty acids were measured with commercially available kitsto quantify each analyte relative to a standard reference. Endogenousglucose production is defined as the difference between endogenous totalglucose concentrations and exogenous tritiated glucose levels (3-³Hglucose).

Metabolic tracer studies: A bolus injection of 1-¹⁴C-2-deoxyglucose(2DG) was introduced after 60 minutes of the clamp study to determinetissue-specific glucose uptake. The 2DG can be used as a surrogate forglucose uptake in different organs. Following collection of the finalblood sample, mice are rapidly (˜1-2 min) euthanized with intravenouspentobarbital, and tissues are harvested and frozen with aluminumforceps in liquid nitrogen to be stored for later analyses. Plasmatracer kinetics and tissue content were then used to calculate basal andinsulin stimulated rates of hepatic glucose production, whole-body andtissue-specific rates of glucose uptake from skeletal muscle, heart,white adipose tissue and brown adipose tissue. Tissue is homogenized and1-¹⁴C-2-deoxyglucose-6-phosphate (2DGP) is isolated from 2DG using ananion exchange resin. Changes in rates of glucose turnover and uptakewill reflect organ specific differences in energy expenditure andinsulin sensitivity and will guide future studies and generation oftissue-specific mARC2 KO mice.

Results:

The moderately aged mARC2 KO (C57BL/6N mARC2^(tm2A)) mice are thinnerthan wild type controls maintained on normal chow (FIG. 2A). Theseobservations are in line with the International Mouse PhenotypingConsortium (IMPC) data collected with the same KO mouse at a younger age(3 months old males and females). To confirm mARC2 deletion, transcriptand protein levels were measured in liver homogenates from mARC-2 KO andWT mice using quantitative reverse transcriptase (qRT) PCR (FIG. 2B) andwestern blot (FIG. 2C), respectively. mARC2 is most abundant in mouseliver tissue, however, we routinely measure mARC2 in kidney, lung, andheart tissue. To future explore, we preformed phenotypic and metaboliccharacterization of moderately aged (8 months old) male mice using mARC2KO and wildtype (WT) littermates (N=8 for each group) feed normal chow.

Deleting mARC2 significantly changes body composition (BC), specificallythe mARC-2 KO mice have less body fat. Knocking out mARC2 reduces bodyweight (FIG. 3A) in adult male mice, mARC2 KO mice weigh 22% less thanWT littermate mice. The decrease in body weight (BW) is primarily aresult of reduced fat mass (FIG. 3B), mARC2 KO mice have 16% fat(normalized to body weight) compared to 28% in wildtype littermatecontrols (FIG. 3C). The loss of fat is independent of staturedifferences; no difference in body length or tibia length were observed.Dissection of the mice revealed less abdominal white adipose tissue(WAT). Lean mass was increased in the mARC2 KO (80%) compared to WT(68%) (FIG. 3D).

Metabolic differences are evident in the mARC2 KO. Reduced BW anddecreased fat mass in the mARC2 KO occurred in association withincreased feeding, activity (FIG. 4A, 4B), and energy expenditure (FIG.4C, 4D), suggesting the effect of mARC2 deletion on energy use exceededthe effects of increased feeding. The mARC2 KO mice consume more foodcompared to wildtypes (0.35 mg food g BW⁻¹ vs. 0.21 mg food g BW⁻¹).Metabolic studies have further revealed that the mARC2 KO mice have anincreased energy expenditure (16.190 vs 13.440 kcal kg⁻¹ hr⁻¹) (FIG. 4A,4B); are more active (300.700 vs. 186.130 counts hr-1) (FIG. 4C, 4D).

The mARC2 KO mice have improved glucose tolerance and insulinsensitivity. Fasting glucose was lower in the 10-month-old male mARC2 KOmice (131.5 mg dL⁻¹) compared to WT mice (197.1 mg·dL⁻¹) (FIG. 5A).Additionally, fasting insulin levels were lower in the KO (0.46 mg·mL⁻¹)relative to WT (1.71 mg mL⁻¹) (FIG. 5B). Intraperitoneal (IP) glucosechallenge demonstrates faster clearance of glucose (28604 vs. 40040 mgdL⁻¹ min.) (FIG. 6A, 6B) and insulin (80.3 vs. 205.6 mg mL⁻¹ min.) (FIG.6C, 6D) in the KO mice compared to WT. Thus, we conclude that mARC2deletion improves energy expenditure, insulin action, and glucosehomeostasis.

Liver histopathology reveals differences in cell structure, possiblyless intercellular lipid accumulation in aged mARC2 KO mice (FIG. 7).While we have not had the opportunity to explore this observationfurther, by staining with lipophilic dyes (e.g. oil red) or markers ofsteatosis, the differences among KO and WT are clear. To elucidate thenature of this observation and determine if mARC2 is a target forinhibiting hepatocyte steatosis.

Published studies have suggested a role for mARC2 in regulation oflipogenesis, however the exact mechanisms at work are not known. Takentogether with our data, we hypothesize that mARC2 is a novel target forthe treatment of obesity and associated diseases (Type 2 diabetes,hepatic steatosis, or NASH).

Moreover, the mARC2 KO mice are prevented from diet induced obesity.(FIG. 9). Twenty-four male C57/BL6N wild-type (WT) and mARC-2 knockout(KO) mice were generated from filial heterozygote breeding. At 9 weeksold, WT and KO littermates were randomly selected to receive a high-fatdiet (HFD) or low-fat diet (LFD), which contained 60% or 10% kcal energyfrom fat, respectively. Of the 24 mice, 14 mice (6 WT, 8 KO) were feedthe HFD and 10 mice (6 WT, 4 KO) were the LFD. Body weights wererecorded weekly. All animal experiments were conducted in accordancewith institutional animal care procedures at the University ofPittsburgh. The longitudinal investigation of body weight in WT andmaRC-2 KO mice revealed that LFD fed mARC-2 KO mice maintained a lowerbody weights than WT littermates over the course of our investigation(FIG. 9). However, the mARC-2 KO mice provided HFD do not maintain a lowbody weight over the entire 20-week experiment. Significant differences(p-value<0.01) in body weight among the WT and KO mice were observedbetween 1 and 10 weeks of HFD exposure, but not at the latter timepoints.

Example 2—Primary Role of Insulin

An intraperitoneal glucose tolerance test was performed on younger, bodyweight matched 12 weeks old mARC2 KO mice, essentially as described inExample 1. In addition to the younger age, these mice have nosignificant differences in body weight, which could be a confoundingvariable in glucose and insulin measurements. Concentrations of glucosewere measured by a glucose oxidase method using a Beckman GlucoseAnalyzer II. Plasma insulin was measured by commercially available Elisaassay, as described in the material and methods section. FIG. 8A showsplasma glucose and FIG. 8B shows insulin levels post-intraperitonealglucose challenge in younger mice. After an overnight fast (time=0min.), no significant differences in plasma glucose or insulin wasobserved (FIG. 8A and FIG. 8B). However, the insulin levels post glucosebolus are significantly higher in the WT mice compared to the mARC2 KOmice over the remaining time points collected in the glucose tolerancetest. While glucose levels are also lower in the mARC2 KO, nosignificance was measured at any of the analyzed time points. Thiscontrasts with aged mice (FIGS. 6A-6D), in which the mARC2 KO miceexhibited improved glucose clearance as well as lower insulin levels. Itis likely that age associated differences in body composition,contributed to the lean mARC2 mice improved glucose clearance relationto the aged WT controls. In conclusion, young mARC2 KO mice haveimproved insulin action, independent of glucose clearance; suggestingthat mARC2 is primarily involved in insulin signaling.

FIGS. 10 to 12 summarize hyperinsulinemic euglycemic clamp and metabolictracer experiments in mice. Body weight was measured pre-catheterimplantation (FIG. 10A), and body weight (FIG. 10B), fat mass (FIG. 10C,left), and lean mass (FIG. 10C, right) were measured post-catheterimplantation. As shown in FIGS. 10A-10C, less fat in seen in mARC2 KOmice at age 12 weeks, but no difference in lean mass was observed. Thisis important because the lean tissue mass is more metabolically activethan the less abundant fat tissues.

Following a 6 h morning fast, hyperinsulinemic euglycemic clampexperiments commenced. In these experiments, radiolabeled glucose(3-³H-glucose) tracer is continuously infused throughout the metabolicclamp experiments, during both “basal” euglycemic and “clamp”hyperinsulinemic euglycemic phase. Blood is collected via tail vein atthe end of basal and clamp phases for plasma fatty acid, insulin, andglucose quantifications (FIGS. 11A-11C). Plasma insulin, glucose, andfatty acid were measured using commercially available assays, asdescribed above in the materials and methods section of this document.During fasting, no difference in plasma fatty acids were detected (FIG.11A) or glucose (FIG. 11C), although basal insulin levels were 70% lessin Marc2 KO compared with WT mice (P<0.05) (FIG. 11B). During insulininfusion, plasma fatty acids levels (FIG. 11A) were significantly lowerin mARC2 KO mice, reflecting improved adipose tissue insulin sensitivity(FIG. 11C; P<0.05). Plasma insulin and glucose levels were approximatelymatched between genotypes during the insulin infusion period of thestudy, although plasma glucose levels were significantly lower in mARC2KO mice (FIG. 11C), despite greater glucose infusion rates (FIGS. 12A &12B).

To assess glucose turnover during hyperinsulinemia, mice received aprimed/continuous infusion of insulin (2.5 mU per kg lean mass per min)and variable infusion of glucose for 120 min in order to match plasmainsulin and glucose levels between groups (FIGS. 11B & 11C). Tail bloodis collected every ten minutes during the clamp phase to measurereal-time glucose levels (FIGS. 12A-12C), such that glucose infusionrates (GIR) can be adjusted in order to match plasma glucose levels at˜120 mg/dl between groups. Increased GIRs therefore reflect increasedinsulin responsiveness. The glucose infusion rate (GIR) required tomaintain euglycemia in the Marc2 KO mice was 2.5-fold greater than WTmice, reflecting increased whole-body glucose utilization and insulinsensitivity. During the last 40 min of the infusion, the GIR was2.5-fold greater in Marc2 KO compared WT mice (FIG. 12B, P<0.01).Consistent with the lack of change in fasting plasma glucose levels,there was no difference in fasting rates of EGP during the basal orclamp phases (FIG. 12C). These data demonstrate that the mARC2 KO micehave much greater rates of glucose utilization than WT controls thatwere due to increased whole-body glucose disposal (P<0.05) and there wasno difference in clamped rates of endogenous (primarily hepatic) glucoseproduction (EGP) (FIG. 12C).

In addition, radioactive 1-¹⁴C-2-deoxyglucose (2DG) tracer wasintroduced during the hyperinsulinemic euglycemic clamp to measuretissue specific glucose transport rates (FIGS. 13A-13E). The glucoseanalog is transported into the cells and converted to1-¹⁴C-2-deoxyglucose-6-phosphate (2DGP) but is not further metabolized.Therefore, intercellular 2DGP is a measure of tissue specific glucoseuptake. Tissue-specific rates of glucose uptake were significantlyincreased in gonadal WAT tissue (1.7×, P<0.05, FIG. 13A), inguinaladipose tissue (2.5×, P<0.01, FIG. 13B) and heart (2.2×, P<0.001, FIG.13C) in mARC2 KO mice. There was a modest, but insignificant increase inbrown adipose tissue glucose transport rates (FIG. 13D) and nodifference in skeletal muscle (gastrocnemius) rates (FIG. 13E).Additionally, the rate of glycolysis was increased in the mARC2 KO mice(FIG. 14), an indication that mARC2 deletion causes improved rates ofglycolysis.

The present invention has been described with reference to certainexemplary embodiments, dispersible compositions and uses thereof.However, it will be recognized by those of ordinary skill in the artthat various substitutions, modifications or combinations of any of theexemplary embodiments may be made without departing from the spirit andscope of the invention. Thus, the invention is not limited by thedescription of the exemplary embodiments, but rather by the appendedclaims as originally filed.

What is claimed is:
 1. A method of treating hepatic steatosis in apatient in need thereof, comprising knocking down expression of amitochondrial amidoxime reducing component (mARC) in a patient, therebytreating hepatic steatosis in the patient, wherein the mARC is mARC2 ormARC1.
 2. The method of claim 1, wherein the mARC mRNA levels arereduced in a cell of the patient.
 3. The method of claim 1, wherein themARC mRNA levels are reduced by administration of a RNAi agent to thepatient.
 4. The method of claim 3, wherein the RNAi specificallyhybridizes to the nucleic acid of SEQ ID NO: 1 or the nucleic acid ofSEQ ID NO:
 2. 5. The method of claim 3, wherein the RNAi ranges from21-33 bases in length and comprises or consists of a sequenceGTGCTCATCTCCATCATTTAT (SEQ ID NO: 5), GATGAACTCCTAATTGGTAGT (SEQ ID NO:6), GAACTCCTAATTGGTAGTGTA (SEQ ID NO: 7), GAGGGATTGACTGAGATCTTA (SEQ IDNO: 8), ACAACAGCAGCAACGATACAT (SEQ ID NO: 9), GATGTGCAGGACGCATGTTAC (SEQID NO: 10), GGACCTACTACTGCCTATCAA (SEQ ID NO: 11), GAAGAGTTATCGCCAGTGTGA(SEQ ID NO: 12), GACCCTTCAGAACGAAAGTTA (SEQ ID NO: 13),GGTGTCTCAATGCTTCAATGT (SEQ ID NO: 14), GGCTGGAAGAATATCCTAGAA (SEQ ID NO:15), or GTGATTTCAGATAGACTACTG (SEQ ID NO: 16).
 6. The method of claim 1,wherein the hepatic steatosis is associated with non-alcoholicsteatohepatitis (NASH).
 7. The method of claim 1, wherein the hepaticsteatosis is associated with Type 2 diabetes.